title: A novel method of detecting alpha-1 antitrypsin deficiency of Z mutant (GAG(342)AAG) in a single PCR reaction using base-quenched probe.
abstract: BACKGROUND
  Alpha-1 antitrypsin (A1AT) is a protease inhibitor that protects the tissues from degradation by neutrophil elastase under certain pathological process. Alpha-1 antitrypsin deficiency (A1ATD) could associate with both lung and liver pathogenicities. Of all the deficiency alleles, Z mutant is the most common variant and causes severe complications. Here, we described a novel and quick method to detect Z mutant using the base-quenched probe technique in only one single PCR reaction.
  
  
  METHODS
  Primers and probe were designed based on the base-quenched probe technique. Two vectors, representing the two genotypes, were constructed as amplification templates for validating the method. The Z mutant (GAG(342)AAG) was analyzed according to the melting curve. Finally, the accuracy was confirmed by direct sequencing.
  
  
  RESULTS
  Z mutant could be accurately distinguished from the wild type. The wild type resulted in high melting temperature (TM) (48.64±1.33°C), while when the Z mutation was present, the TM was shifted to an obvious low TM (41.38±0.9017°C). The sensitivity reached a low of 10(3) copies of template DNA with a clear melting valley and a complete concordance occurred between this method and the direct DNA sequencing.
  
  
  CONCLUSIONS
  The present described method is simple, quick and economic as well as suitable for large-scale genotyping studies and clinical testing of Z mutant in patients with emphysema and cirrhosis.
authors.affiliation: Comprehensive Laboratory, Changzhou Key Lab of Individualized Diagnosis and Treatment Associated with High Technology Research, The Third Affiliated Hospital of Soochow University, Changzhou 213003, China.
authors.forename: Li
authors.initials: L
authors.lastname: Qin
authors.forename: Guanghua
authors.initials: G
authors.lastname: Luo
authors.forename: Jun
authors.initials: J
authors.lastname: Zhang
authors.forename: Ning
authors.initials: N
authors.lastname: Xu
mesh_terms: D001483:Base Sequence; D004252:DNA Mutational Analysis / Q000379:methods*; D005453:Fluorescence; D005838:Genotype; D006801:Humans; D009154:Mutation / Q000235:genetics*; D029424:Pulmonary Disease, Chronic Obstructive / Q000097:blood / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D013997:Time Factors; D044366:Transition Temperature; D019896:alpha 1-Antitrypsin Deficiency / Q000097:blood / Q000175:diagnosis / Q000235:genetics*
keywords: Alpha1-antitrypsin deficiency; PIZ mutant; Single nucleotide polymorphism